Unraveling the dynamic oxidative processes after ischemia/reperfusion in the heart using new proteomics approaches

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Facultad de Medicina Departamento de Bioquímica

Programa de Doctorado en Biociencias Moleculares

Unraveling the dynamic oxidative processes after ischemia/reperfusion in the heart using

new proteomics approaches

TESIS DOCTORAL

Celia Castañs García

Graduada en Bioquímica

Codirectores de tesis:

Prof. Jesús Vázquez Cobos Dr. Inmaculada Jorge Cerrudo

Madrid, 2019

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Jesús Mª Vázquez Cobos, DOCTOR en Ciencias Químicas por la Universidad Autónoma de Madrid y Profesor de Investigación del Consejo Superior de Investigaciones Científicas; actualmente Full Professor en el Centro Nacional de Investigaciones Cardiovasculares (CNIC) de Madrid; e Inmaculada Jorge Cerrudo, DOCTORA en Ciencias por la Universidad de Córdoba.

CERTIFICAN que,

Doña Celia Castañs García, Graduada en Bioquímica por la Universidad Complutense de Madrid, ha realizado bajo su dirección el trabajo de investigación titulado:

“Unraveling the dynamic oxidative processes after ischemia/reperfusion in the heart using new proteomic approaches”

y consideran que el trabajo realizado reúne todas las condiciones requeridas por la legislación vigente, así como la originalidad y calidad científicas necesarias, para poder ser presentado y defendido con el fin de optar al grado de Doctor, con Mención Internacional, por la Universidad Autónoma de Madrid.

Y para que así conste y surjan los efectos oportunos, firmo el presente certificado en

Madrid, a 27 de febrero de 2019 Madrid, a 27 de febrero de 2019 Director de la Tesis Doctoral Co-director de la Tesis Doctoral Dr. Jesús Mª Vázquez Cobos Dra. Inmaculada Jorge Cerrudo

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Esta tesis doctoral ha recibido financiación del Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario

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“Y cuando la tormenta de arena haya pasado, tú no comprenderás cómo has logrado cruzarla con vida. ¡No! Ni siquiera estarás seguro de que la tormenta haya cesado de verdad. Pero una cosa sí quedará clara. Y es que la persona que surja de la tormenta no será la misma persona que penetró en ella.

Y ahí estriba el significado de la tormenta de arena.”

Haruki Murakami

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Agradecimientos

Me gustaría agradecer a Jesús Vázquez la oportunidad brindada para iniciar esta aventura, así como toda su ayuda, energía y disposición. Gracias también a mi codirectora, Inma, por toda la ayuda durante la elaboración de la tesis y a mi tutor de la UAM, Miguel, por su apoyo en cada trámite.

Tengo que agradecer a todos los trabajadores del CNIC por toda su ayuda durante estos casi 5 años, desde la cafetería a RRHH, y en especial a nuestros colaboradores del laboratorio de Borja Ibañez.

Muchas gracias a todos mis compañeros del Laboratorio de Proteómica y de la Unidad de Proteómica: los que están y los que se fueron, los de la cueva, el laboratorio, la primera sur, la zona postdoc, los establos y las oficinas. Todos los miembros de esta gran “familia” han dejado su marca en el desarrollo de esta experiencia. En especial me gustaría agradecer a Marco, Juan Antonio y Navratan por su generosidad y amistad.

Probablemente el mejor resultado de esta tesis sea un grupo de personas que aparecieron en este viaje en distintas estaciones para quedarse una vez concluido: Aleks, Ricardo, Ile, Fer, Jesús L, Rocío, Alessia, Diego e Irene. Con ellos he vivido las más surrealistas e inolvidables aventuras dentro y fuera del CNIC, en España y en el extranjero; y realmente no sé qué hubiera sido de mí sin su amistad, su ayuda, su buen humor y sus consejos. En especial no tengo palabras suficientes para agradecer a Aleksandra todo lo que ha hecho por mí en su papel de amiga, de compañera y de mentora.

Gracias a mi familia por su amor y apoyo incondicional, y a mis amigos por ser prácticamente mi familia.

Gracias, Jaime, por obligarme a construirme unas alas en la caída.

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Summary

After an episode of coronary artery occlusion, myocardial response to reperfusion does not remain stable during time. Although there are many relevant proteomic changes taking place in the tissue, systematic studies are lacking on the protein modification changes occurring in the post-reperfused myocardium along the first week after I/R. In this Doctoral Thesis we present the application of global PTMs analysis combined with the mapping of redox-active thiols in myocardial tissue proteins to study the oxidative and molecular dynamic changes after I/R.

After ischemia, blood flow restoration generates an initial oxidative damage, generated in the mitochondria, that we detected at 20 minutes after reperfusion. This initial and intracellular oxidative wave consisted on irreversible monooxidations and affected intracellular proteins essential for cardiac function.

Later, starting at 2h and peaking at 24h after reperfusion, immune cells, including neutrophils, are recruited to the lesion site as part of the cardiac inflammatory process. We detected an increase in neutrophil’s granule peroxidases in the tissue, as well as a second oxidative wave consisting on irreversible dioxidations and trioxidations and also Cysteine reversible oxidation, in extracellular and membrane proteins. Towards the end of the first week and with the start of the repair and fibrotic phase, we detected an increase in collagen proline-hydroxilations as well as lipid peroxidation accumulation in the tissue.

Additionally, these results were validated in other animal models.

This doctoral thesis provides the first systematic understanding of the timeline of the different oxidative processes during the first week after I/R. This study comprises the deep study of the posttranslationally modified peptidome, the redoxome and the proteome in a highly translational pig model, and might have implications on the definition of the disease progression and in the prediction and future approaches of the disease.

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Resumen

Después de un episodio de oclusión de una arteria coronaria, la respuesta del miocardio a la reperfusión no permanece estable en el tiempo. En este sentido, aunque estudios previos han mostrado cambios importantes en el proteoma durante este periodo, faltan estudios sistemáticos sobre los cambios en modificaciones de proteínas que ocurren en el miocardio durante la primera semana después de la reperfusión. En esta Tesis Doctoral mostramos un análisis global de PTM combinado con el estudio del proteoma redox y la biología de sistemas para revelar los cambios moleculares que tienen lugar en la semana posterior a la I/R.

Después de la isquemia, la restauración del flujo sanguíneo genera un daño oxidativo inicial, originado en las mitocondrias, que detectamos 20 minutos tras la reperfusión. Esta onda oxidativa inicial se caracteriza por monooxidaciones irreversibles que afectan proteínas intracelulares esenciales para la función cardíaca. Más tarde, desde las 2 horas y, especialmente a las 24 horas tras la reperfusión, las células del sistema inmune se infiltran en la lesión como parte del proceso inflamatorio. De esta forma detectamos un aumento en las peroxidasas de los gránulos de neutrófilos en el tejido, así como una segunda onda oxidativa consistente en dioxidaciones y trioxidanciones irreversibles y oxidaciones reversibles en cisteínas, afectando proteínas extracelulares y de membrana. Hacia el final de la primera semana y dando lugar a la fase de fibrosis, detectamos un aumento en las hidroxilaciones de prolina de colágeno y una acumulación de lípidos peroxidados en el tejido.

Esta tesis doctoral proporciona la primera comprensión sistemática del transcurso de los diferentes procesos oxidativos durante la primera semana después de la I/R. Estos resultados fueron validados, además, en otro modelo animal y comprenden un estudio profundo del peptidoma modificado postraduccionalmente, el redoxoma y el proteoma en un modelo de cerdo altamente traslacional, pudiendo tener implicaciones en la definición del progreso de la enfermedad y en la predicción y enfoque futuro de la misma.

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Abbreviations

TERM DESCRIPTION

AMI Acute myocardial infarction

Asn Asparagine

Asp Aspartic Acid

C Cysteine

CM Cardiomyocyte

CMR Cardiac magnetic resonance

CNIC Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III

CVD Cardiovascular disease

Cys Cysteine

D Aspartic Acid

Da Dalton

ECM Extracellular Matrix

EGC Electrocardiogram

ETC Electron Transport Chain

F Phenylalanine

FA Fatty Acid

FC Fold change

FDR False discovery rate

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Abbreviations

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GO Gene Ontology

HCA Hierarchical Clustering Analysis

HNE Hydroxynonenal

HPLC High performance liquid chromatography

i.v. Intravenous

I/R Ischemia/reperfusion

ICAT Isotope-coded affinity tag

IgG Immunoglobulin G

K Lysine

LAD Left anterior descending

LF Label Free

Ly6G Lymphocyte antigen 6 complex locus G6D

Lys Lysine

Mat&Met Materials and Methods

MDA Malondialdehyde

MI Myocardial infarction

MPO Myeloperoxidase

MPTP Mitochondrial permeability transition pore

MS Mass spectrometry

MS/MS Tandem mass spectrometry

N Asparagine

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Abbreviations

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nanoLC-MS/MS Nano-liquid chromatography-tandem mass spectrometry

NOR Non-reperfusion

NSTEMI non-ST-elevation myocardial infarction

P Proline

PANTHER Protein ANalysis THrough Evolutionary Relationships

PC Pre-conditioning

PCA Principal Components Analysis

PCI Percutaneous coronary intervention

Phe Phenylalanine

ppm parts-per-million

Pro Proline

PSM Peptide-Spectrum Match

PTM Postranslational modifications

ROS Reactive oxygen species

rpm revolutions per minute

SBT Systems biology triangle

SD Standard Deviation

SIL Stable isotope labeling

STEMI ST-elevation myocardial infarction

TCA Tricarboxylic acid cycle

TMT Tandem Mass Tag

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Abbreviations

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Trp Tryptophan

Tyr Tyrosine

W Tryptophan

WB Western Blot

WSPP Weighted spectrum, peptide and protein model [Statistical Model]

Y Tyrosine

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Introduction

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Introduction

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Acute myocardial infarction: relevance

Acute myocardial infarction (AMI) is an event of myocardial necrosis caused by an unstable ischemic syndrome [1]. In the clinic, patients with AMI usually present with chest pain and it is eventually diagnosed in the basis of electrocardiogram (ECG), biochemical tests, imaging, pathological and clinical evaluation [2, 3].

In most developed countries, AMI is a significant clinical burden. Heart attacks and strokes are responsible of the 80% of cardiovascular disease (CVD) deaths. Individuals at risk of CVD may demonstrate raised blood pressure, glucose, and lipids as well as overweight and obesity. Although its high incidence, there has been a significant decrease in cardiovascular mortality since the 1970s due to advances in the diagnosis and management of AMI [2, 4]. Today, with percutaneous coronary intervention (PCI) and stenting, antithrombotic therapy and the routine use of complementary medical treatment, in-hospital mortality from AMI has significantly decreased [2, 5].

AMI can be classified into two categories based on the ECG manifestation of ST-elevation: non-ST-elevation myocardial infarction (NSTEMI) and ST- elevation myocardial infarction (STEMI)[4]. The major cause of STEMI is coronary atherosclerosis with luminal thrombus, which accounts for more than 80% of all infarcts[6]. STEMI most frequently arises with a rupture of an atherosclerotic plaque, which exposes the blood to thrombogenic lipids and leads to activation of platelet and clotting factors, generating a thrombotic formation that completely occludes a major epicardial coronary vessel [2-4]. Artery occlusion produces a life-threatening ischemic damage that requires urgent intervention. Extending the period of acute myocardial ischemia for more than 20 minutes causes massive necrotic cell death within the infarcted tissue [7, 8].

Thus, the most effective therapeutic intervention is myocardial blood reperfusion using thrombolytic therapy or primary percutaneous coronary intervention to rescue the ischemic myocardium [7].

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Introduction

26 I/R injury and Edema

The degree of necrosis in the heart tissue produced by myocardial infarction has been shown to be critical to determine subsequent chronic complications that infarct survivors suffer from [9]. Hence, there is an urgent need to develop novel therapies to minimize infarct size, which would result in better long term heart performance with fewer adverse clinical events, reduced mortality, and a significant relief of the huge socioeconomic burden [10, 11].

Blood flow restoration always accompanies the treatment for myocardial infarction, in a process known as reperfusion, which is the only available treatment to stop the progression of ischemic damage during an AMI[6].

Although early reperfusion is the best strategy to stop the damage progression, it paradoxically produces an additional stress to the myocardium (reperfusion injury), which may account for up to 40% of infarct size [12, 13]. These two processes together constitute what is known as I/R injury.

Myocardial infarction and infarct size is revealed by a manifestation of different forms of cardiomyocyte (CM) cell death: necrosis, apoptosis, autophagy, and necroptosis. During ischemia, the anaerobic glycolysis increases the influx of sodium ions (Na⁺) through the Na⁺/hydrogen ion (H⁺) exchanger due to developing acidosis. Moreover, intracellular Na⁺ accumulation is increased by the inhibition of Na⁺/ potassium ion (K⁺) ATPase due to the lack of available ATP molecules [14]. The subsequent exchange of Na⁺ for Ca²⁺ by reverse mode operation of the sarcolemmal Na⁺/Ca²⁺ exchanger induces intracellular Ca²⁺

overload. After reperfusion, the rapid normalization of pH in the context of elevated cytosolic Ca²⁺ induces subsequent release and reuptake of Ca²⁺ into the sarcoplasmic reticulum, causing uncontrolled hypercontracture [15, 16]. The high cytosolic concentrations of Na⁺ and Ca²⁺ also results in intracellular edema when extracellular osmolarity is abruptly normalized by reperfusion. Furthermore, the opening of the mitochondrial permeability transition pore (MPTP) appears to be decisive for necrosis, apoptosis, and necroptosis, and mitochondria are also decisive in mitophagy/autophagy.

While the contribution of reperfusion injury to final infarct size has been disputed in the past, today it is accepted that reperfusion alters post-myocardial

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Introduction

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infarction (MI) repair and appears to produce dynamic molecular changes that evolve within the first week after the ischemia/reperfusion (I/R) onset [17-22].

The typical microscopic features of reperfused myocardial infarction include:

contraction band necrosis, nuclear dissolution, mitochondrial swelling and disruption, CM membrane disruption, microvascular destruction, interstitial hemorrhage, and inflammation [11].

Under basal conditions, water is the main component of healthy heart tissue, since the water content in the myocardium is stable and, mostly, intracellular, with a very small part as interstitial component in the extracellular matrix. During myocardial infarction, edema initially occurs as swelling of CM during the early stages of ischemia [23]. Then, myocardial edema is significantly increased after restoring blood flow to the ischemic region due to reperfusion.

This increase associated with reperfusion appears to be due to an increase in cellular swelling [24] and, more importantly, interstitial edema [25] due to reactive hyperemia and leakage of damaged capillaries when hydrostatic pressure is restored after reperfusion [26, 27].

It has been believed for many years that the intense edematous reaction that is occurred early after MI in the post-ischemic region remained stable for at least a week. [28, 29]. Only recently, post-I/R myocardial edema has been shown to follow a consistent bimodal pattern in animal model and in MI patients [17, 19]. In these studies, serial cardiac magnetic resonance (CMR) imaging shows that tissue composition changes dynamically during the first week after infarction as initial edema was detected within the first 3h after the onset of reperfusion, dissipated at 24h, and was followed by a delayed wave that reappeared 4-7 days after infarction [17, 19].

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Figure 1. Mechanisms Underlying the Bimodal Edema Phenomenon After Myocardial Ischemia/Reperfusion. Two distinct waves of edema emerge within the first week after ischemia/reperfusion (I/R) because of different pathophysiological processes. The initial wave, appearing abruptly on reperfusion, is a direct consequence of the reperfusion process itself, whereas the deferred wave, appearing progressively days after I/R, is mainly caused by tissue healing processes. (A) Reperfusion is associated with a very abrupt edematous reaction that separates myocardial fibers from each other, resolving by day 1. (B) Boxes represent water content measurements in pigs; blue ¼ regular I/R protocol; salmon ¼ permanent coronary occlusion (nonreperfused myocardial infarction; initial edema wave abrogated, deferred wave significantly attenuated); green ¼ I/R plus steroid therapy (deferred wave significantly attenuated). The lines show cardiac magnetic resonance T2 relaxation time course in the ischemic myocardium; blue ¼ first week after infarction (2 peaks closely track the water content changes); salmon ¼ nonreperfused myocardial infarction (both waves of cardiac magnetic resonance–evaluated edema significantly attenuated); and green ¼ I/R plus steroid therapy (continuous line after initiation of therapy).

(Source Fernández-Jiménez, R. et al. 2015 [18]).

Moreover, our group recently described detailed protein expression changes that take place in both the ischemic and remote myocardium during the first week after infarction in a pig model [20]. This study revealed a highly coordinated, multimodal pattern of functional protein alterations in both

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Introduction

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myocardium regions in the early stages post I/R. First, early reperfusion can be characterized by an abrupt edematous response [17, 18] and a rapid and profound acute inflammatory reaction [13, 30]. Intracellular, mitochondrial and sarcolemmal Ca²⁺ levels increase strongly upon reperfusion, resulting in hypercontraction of the CM and opening of the MPTP, the hallmark of reperfusion-induced CM death [31]. The previous proteomic characterization performed by our group [20] has showed the temporal dynamics of hundreds of protein changes involved in inflammation, wound healing, and reactive oxygen species as early as 120 minutes after the reperfusion procedure. This comprehensive study identified a rich repository of early-response proteins directly related to reperfusion injury that indicates an early event after reperfusion.

Later, at 4 and 7 days after reperfusion, coordinated protein changes in the ischemic myocardium revealed the activation of protein biosynthesis, the synthesis of collagen and interstitial proteins, and the formation of new vessels, along with the depletion of muscle and mitochondrial proteins. These processes reflected a phenotypic change in infarcted myocardial tissue characterized by the replacement of necrotic myocytes, rich in mitochondria, by collagen, extracellular matrix proteins, and fibroblasts with low mitochondrial content, suggesting a later event of repair and remodeling. This study also demonstrated that I/R significantly impacts the remote myocardium, inducing transient molecular alterations of sarcomere components, coinciding with CMR-detected transient stunned myocardium [19, 32], which reverts to control levels by 7 days after reperfusion.

The exact molecular and cellular events that occur during this very dynamic early post-I/R phases are still largely unknown. Experimental procedures for the study of I/R response using reperfused and non-reperfused MI models and preconditioning strategies can accurately reflect healing processes. Thus, experimental models on animals are of crucial importance in I/R research, as there are only a limited number of in vitro models [32, 33]. Much of the prevailing knowledge about I/R and the molecular modifications involved comes from studies performed with animal models, mainly rodents [34-37] or pigs [38]. The similarity in size, physiology, and in heart development and disease

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Introduction

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progression make the swine an ideal model for I/R research and its direct translation of results into humans [39].

Cardioprotective interventions to decrease I/R injury

Although the mortality of STEMI in Western countries has declined in the past decades, the still high incidence encourages the seek for new therapies focused on reducing the I/R injury.

First, it was reported [40] that brief cycles of ischemia and reperfusion performed before a prolonged coronary artery occlusion with reperfusion could substantially reduce final infarct size in dogs to 25%, what was referred as

“ischemic pre-conditioning”. Unfortunately, the unpredictability of coronary artery occlusion event in STEMI patients means that ischemic pre-conditioning cannot be applied in this clinical setting, but it could have an important role in planned procedures like cardiac surgery [41] or in diabetic and non-diabetic patients with stable coronary artery disease [42].

Later, “ischemic post-conditioning” was described in 2003 [43], showing that brief episodes of ischemia and reperfusion performed immediately after reperfusion following ischemia could reduce final infarct size in dogs by 30% to 40%. This intervention is applied after flow restoration; consequently, it has no connection to the duration of ischemia and must be related to the prevention of events occurring after reperfusion. In other study in 2005 [44], it was also demonstrated that ischemic post-conditioning could reduce infarct size in STEMI patients. Another form of myocardial conditioning well described in animal models is remote ischemic conditioning: conditioning performed in a distant organ [45]. These promising results in were followed by clinical studies, and this might potentially translate into clinical setup [46].

Oxidative PTMs and I/R injury

While reperfusion of ischemic tissue is essential for survival, it also initiates oxidative damage through generation of reactive oxygen species (ROS).

This event has been consistently demonstrated by measuring free radical generation in perfused rabbit hearts by electron paramagnetic resonance

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spectroscopy [47], in reperfused rat hearts by electron spin resonance spectroscopy [48], in Langendorff guinea pig hearts through fluorescent indicators [49] and measurement of superoxide-derived free radicals [50], showing a burst of ROS generation on the start of reperfusion, peaking from 4 to 10 minutes. Mitochondria are considered to be a major source of ROS as well as a major target for ROS damage due to the role of mitochondrial electron transport chain (ETC) as one of the primary sources of ROS in the cell [51, 52]. Additionally, accumulation of succinate and its re-oxidation after reperfusion has been pointed out as the main contributor to extensive ROS peaking at 10-15 min after reperfusion at mitochondrial complex I in in vivo and Langendorff mice hearts [53].

Postranslational modifications (PTMs) are well known to have a pivotal role in the control of signaling cascades and protein structure and functionality.

Some studies have tried to shed some light into the effect in proteins of mitochondrial disease or initial ROS production 5 minutes after reperfusion studying the thiol redox status in mitochondria using ICAT reagents [54, 55].

Other approaches have made it possible to measure protein oxidation and s- nitrosylation of Cys after I/R in Langendorff mice hearts using Resin-Assisted Capture and showing an increase in protein oxidation 5 minutes after reperfusion [56]. In this line, GELSILOX [57] has been presented as a technology that allows the simultaneous quantification of proteins and of reduced and oxidized Cys sites in the same experiment, allowing the identification of Cys-reversible oxidation ROS targets. This method revealed an increase in Cys-oxidation 5 minutes after reperfusion in Langendorff rat hearts and isolated mice mitochondria and made it able to identify the specific site of oxidation [58]. Moreover, it showed that ischemic preconditioning substantially reduces the extent of Cys-oxidation and that isolated mitochondrial preconditioning reduces mitochondrial ROS production [57, 58], suggesting that the mitochondria has all the machinery necessary for ROS production and preconditioning protection. All these studies consistently demonstrate that initial mitochondrial ROS production caused by reperfusion is having an effect in protein modification, while the global extent or the nature of this damage remains unknown.

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Figure 2. I/R induces mitochondrial ROS. During I/R, the activity of ETC complexes is reduced by an extraordinary increase in ROS, which is a result of succinate accumulation under ischemia.

Upon reperfusion, succinate is rapidly oxidized resulting in a burst of ROS, probably mediated by a reverse electron transport from complex II to complex I. The increase of ROS together with an I/R-induced calcium influx into mitochondria opens the MPTP, further increasing ROS formation, decreasing ETC activity, and finally provoking cell death. (Source Fuhrmann, D. C. et al, 2017 [59]).

Mitochondrial ROS production after I/R has been considered as an initial damage burst that gradually recovers during the next few hours after reperfusion [47, 48, 50, 60, 61]. In contrast, the bimodal nature of edematous myocardial response [17-19], and the previous molecular characterization of the dynamic protein changes during the first week after reperfusion [20] suggests that there are more than one initial oxidative event. Supporting these assumptions, some authors have showed an increase in 4-HNE and MDA lipid-peroxidation markers peaking as late as 24h after reperfusion [62, 63]. Unlike mitochondrial ROS, these studies suggest the role of NADPH oxidases as major sources of later ROS production in the heart and mediating myocardial I/R injury. Additionally,

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authors described that this peak of 4-HNE and MDA can be markedly ameliorated at 4h after reperfusion in mice with the administration of interleukin-37 [63]. Interleukin-37 is a newly identified member of the IL-1 family, and functions as a fundamental inhibitor of innate immunity and inflammation. It has been thoroughly reviewed that early after reperfusion, tissue injury and necrosis initiate an inflammatory phase [8, 64]. It consists of an intense inflammation and the dynamic recruitment of neutrophils, monocyte/macrophages, dendritic cells, and lymphocytes. Furthermore, it has been suggested that part of the myocardial ROS could be generated by infiltrated leucocytes, especially activated neutrophils in the injured myocardium [65], but the relation between myocardial injury and neutrophil infiltration remains unknown. These results suggests that the innate immune and inflammatory responses are playing a major role in later I/R injury, which might be part of a different oxidative event in the myocardium.

Very recently, Aleksandra Binek, from our group, described in her PhD thesis [66] an analysis of the Cys redox status in ischemic and remote tissue in a pig model at 2h, 24h, 4 days and 7 days after I/R. In this study, the authors showed that, while no apparent increase in thiol-redox levels was detected in the remote tissue, data from pooled (n=5) protein extracts revealed a very clear transient increase in reversible oxidation of Cys sites in the infarcted area, peaking at 24h post-reperfusion (Figure 3) but starting at 2h after reperfusion.

The previous results from our group [20] described that early after reperfusion, the ischemic myocardium showed a coordinated increase in the expression of proteins involved in acute-phase response signaling, response to wounding, wound healing, blood cell adhesion, and production of nitric oxide and reactive oxygen species. These processes find its peak at 24h and remain activated during the first week of I/R in agreement with the presented Cys-oxidation increase.

These results also confirmed that there are relevant oxidative processes going on in the ischemic tissue before 120 minutes after reperfusion.

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Figure 3 Cysteine reversible oxidation in the infarcted and remote tissues during the first week after MI. Oxidized Cys-peptides changes along time course (2h-7 days) in the remote and ischemic tissue. Total population of oxidized cysteines Zp values are reported as the log2-fold changes expressed in units of standard deviation around the averages with respect to controls. Data from Aleksandra Binek PhD Thesis [66].

Despite all these advances, to date there have been no systematic studies on the protein modification changes occurring in the post-reperfused myocardium along time after I/R. Considering that it has been showed that myocardial response to reperfusion does not remain stable during the first week after I/R [17-19] and, additionally, there are many relevant proteomic and redox changes taking place in the tissue [20], local and time-restricted studies remain insufficient. Ample temporal information about the extent of I/R-induced proteome-wide modifications, their nature and pathophysiological source is needed to establish timely and properly addressed administration of antioxidant therapies, which would in turn more effectively minimize the infarct size.

-10 -5 0 5

10 Zp > 1.5

Zp < -1.5

2h 24h 4day 7day

Zpdistribution (log2Fold Change I/R vs. baseline)

-10 -5 0 5 10

2h 24h 4day 7day

Remote tissue Zp > 1.5 Ischemic tissue

Zp < -1.5

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Introduction

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MS based proteomics and PTM analysis

Enormous efforts are made to get a complete glimpse of PTMs in biological systems, but we are still far away from understanding and unravelling the combinatorial complexity of PTMs. In spite that MS is without any doubt the most powerful technique for PTMs identification and quantitation, and that a large number of relevant PTMs can be directly characterized in biological samples without prior enrichment, PTMs analysis in high throughput proteomics experiments is rarely performed in the routine. This is because database search engines used in proteomics need the exact change in mass and knowledge about the modifiable sites, and only a few PTMs (typically less than four) can be identified at the same time, due to the exponential increase in searching space.

Hence, PTMs have to be searched consecutively, and analyzing only the most common modifications in just one high throughput experiment becomes so time- consuming that it is poorly affordable in the practice. A related recently reported problem is that accurate PTMs identification makes it necessary to compute the local FDR (False Discovery Rate; i.e. in the population of peptides with the modification), which may be considerably different from the global FDR (calculated with all the peptides) [67]. Thus, another currently unresolved challenge in the field is the identification of large number of PTMs without losing the identification accuracy provided by the local FDR.

Recently, a novel mass-tolerant database searching method has been proposed capable of identifying a considerable proportion of PTMs in high throughput experiments [68], revealing that many of the unassignable spectra in proteomics data represent peptides with post-translational modifications. Later report showed that mass-tolerant database search can be performed at faster speeds using fragmentation-ion indexing algorithms [69]. Although these methods are not able to identify the modified sites and misses many modifications, they opened the possibility of real hypothesis-free high- throughput systematic study of PTMs by MS.

On the other hand, quantification of PTMs is also very challenging. First, PTM-containing peptides are often low abundant. Second, the very high dynamic range of protein concentrations in a cell presents serious difficulties for the

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Introduction

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quantification of PTMs. Third, PTM quantification not only has to cover the proteins change in abundance but also the determination of the modification site.

Many algorithms [70, 71] have provided a promising way for quantification of proteins and can be directly extrapolated to PTMs. However, these methods lack a generic quantification model for PTMs and a general statistical model for the analysis of data of this nature.

Our group recently reported Comet-PTM [72], an improved mass-tolerant database search engine for comprehensive identification of PTMs, which enhances the coverage attained by other existing algorithms, assigns the modified residues with an estimated accuracy of 85%, and enables their quantitative analysis through an integrated statistical model. Comet-PTM takes into account the mass shift produced by the modification in the fragmentation series, producing the same score as a narrow-tolerance database search using the same mass increment as a variable modification. This method also introduces a conservative three-layered approach to control the FDR of peptide identification:

local, peak and global FDR.

Many of the quantitation limitations can be dealt with in a single statistical framework previously developed in our laboratory called weighted spectrum, peptide, and protein model (WSPP) [73]. WSPP model allows a systematic comparison and establishes a validity of null hypothesis at each one of the levels (spectrum, peptide and protein), which allows a more accurate modelling of the heterogeneous variance at all levels. Therefore, the statistical approach applied by this method enables quantitative analysis of the modified peptidome within the same framework used to analyze the unmodified proteome; this approach thus allows coherent interpretation of all the results and opens the way to integrated systems-biology analysis. Along with WSPP model, another model developed by our group called systems biology triangle (SBT) allows the study of protein coordination by pairwise quantitative proteomics [74].

Thus, this revolutionary PTM approach could be coupled with the dynamic thiol redox status analysis using differential alkylation technology [57] to study labile Cys modifications and irreversible protein modifications at different time- points after I/R in pig models, as well as the proteome global changes.

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Objectives

The extent of ischemic injury produced by acute myocardial infarction (AMI) is critical to determine the subsequent treatment for the chronic complications that infarct survivors suffer. Despite the efforts devoted to study molecular mechanisms that regulate cardiac remodeling after AMI, it is mandatory to understand the crucial molecular events in order to improve the prediction of disease evolution after ischemia/reperfusion (I/R). Previous thiol oxidation, proteomics and physiopathological studies, as well as the vast knowledge of this area of research, have suggested the presence of more than one systemic molecular event in the ischemic myocardium during the first week after I/R.

Therefore, in this Doctoral Thesis we have pursued the following objectives:

1) Study the global thiol redox proteome events in early time points after AMI, from minutes to days after reperfusion, in the ischemic area of heart using a large pig model of I/R.

2) Identify proteome-wide, irreversible post-translational modifications and quantify their changes after AMI and study their relation to protein changes and chemical alterations.

3) Analyze the effects of cardioprotective treatments in protein modifications in the infarcted tissue.

4) Study the molecular impact of AMI on the remote myocardium in the early time points after I/R.

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Materials and Methods

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Materials and Methods

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Study Design of the time-course after I/R

Pig I/R experiments were performed on a total of 40 castrated male Large- White pigs weighing 30 to 40 kg and were approved by the Institutional Animal Research Committee. The study design is summarized in Figure 5 in the Results section. In 36 pigs reperfused acute myocardial infarction (AMI) was induced experimentally by closed-chest 40-minute mid left anterior descending coronary artery occlusion. These pigs were sacrificed at 20 minutes (n=4), 40 minutes (n=4), 80 minutes (n=4), 2 hours (n=4), 6 hours (n=4), 12 hours (n=4) and 24 hours (n=4) after reperfusion. Four animals were sacrificed at 120 minutes after artery occlusion without reperfusion (120min NOR); and four animals were subjected to ischemic preconditioning prior to I/R and sacrificed at 24h (24h PreC). The other four pigs were sacrificed with no intervention other than baseline CMR, and served as controls. CMR scans were performed at every follow-up stage until sacrifice by Borja Ibañez’s Laboratory at CNIC. Animals were immediately euthanized after the last follow-up CMR scan, and transmural myocardial tissue samples from ischemic and remote areas were rapidly collected for proteomics evaluation. Based on anatomical correlates and standard left ventricle segmentation, those areas from mid-apical ventricular short axis slices matching regional contractility analysis were selected for sampling collection. For mice I/R experiments, we used wild-type 8–13-week-old C57BL/6 mice. The study design is summarized in Figure 32 in the Results section. Three mice were sacrificed with no intervention and served as controls. In the other mice, surgically-induced MI was performed as previously described [75] and sacrificed at 10 minutes (n=3), 20 minutes (n=3), 40 minutes (n=3), 80 minutes (n=3), 2 hours (n=3), 6 hours (n=3), 12 hours (n=3), 24 hours (n=3) and 48 hours (n=3) after reperfusion.

Animal Experimentation

Animal experimental procedures related to the myocardial infarction pig and mouse models were carried out and kindly provided by the members of the Translational Laboratory for Cardiovascular Imaging and Therapy under the supervision of the principal investigator Borja Ibañez at CNIC.

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42 Myocardial infarction procedure in pig

The I/R protocol has been detailed elsewhere [17]. Anesthesia was induced by intramuscular injection of ketamine (20 mg/kg), xylazine (2 mg/kg), and midazolam (0.5 mg/kg) and maintained by continuous intravenous infusion of ketamine (2 mg/kg/h), xylazine (0.2 mg/kg/h), and midazolam (0.2 mg/kg/h).

Animals were intubated and mechanically ventilated with oxygen (fraction of inspired O2: 28%). Central venous and arterial lines were inserted, and a single bolus of unfractioned heparin (300 IU/kg) was administered at the onset of instrumentation. The left anterior descending coronary artery, immediately distal to the origin of the first diagonal branch, was occluded for 40 minutes with an angioplasty balloon introduced via the percutaneous femoral route using the Seldinger technique. Balloon location and maintenance of inflation were monitored angiographically. After balloon deflation, a coronary angiogram was recorded to confirm patency of the coronary artery. A continuous infusion of amiodarone (300 mg/h) was maintained during the procedure in all pigs to prevent malignant ventricular arrhythmias. In cases of ventricular fibrillation, a biphasic defibrillator was used to deliver non-synchronized shocks.

Methods for I/R in mice

Male 8–12-week-old mice were subjected to 40 min occlusion of the LAD coronary artery followed by 10 min, 20 min, 40 min, 80 min, 2 hours, 6 hours, 12 hours, 24 hours or 48 hours of reperfusion. The I/R procedure was performed as previously described [75]. For the LAD procedure, mice were intra-peritoneal anesthetized with ketamine (60 mg/kg), xylacine (20 mg/kg), and atropine (9 mg/kg). Fully asleep animals were intubated and temperature controlled throughout the experiment at 36.5 °C to prevent hypothermic cardioprotection.

Thoracotomy was then performed and the LAD was ligated with a nylon 8/0 monofilament suture for 40 min. The electrocardiogram was monitored to confirm total coronary artery occlusion (ST-segment elevation) throughout the 40 min ischaemia.

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43 Neutrophil depletion procedure

Neutrophil depletion was performed as described before [76]. Briefly, Neutrophils were depleted in C57BL/6 male by i.v. injection of 50 μg anti-mouse Ly6G at 24h and 48h prior to I/R and sacrificed 24h after reperfusion (n=3). The study design is summarized in Figure 36 in the Results section. A second group consisting on three mice were injected with IgG Antibody as vehicle and subjected to I/R and 24h of reperfusion. A third group of three mice with no injection were also sacrificed 24h after reperfusion (n=3). Nine additional mice from the three previous groups were sacrificed without I/R intervention: control without injection (n=3), IgG injection (n=3) and Ly6G injection (n=3).

Proteomics analysis

Proteomics protocol is schematized in Figure 4.

Figure 4 Proteomics protocol scheme. For proteomic analysis, remote and ischemic tissue samples from pig and whole hearts from mice were processed for protein extraction, tryptic digestion, differential Cys labelling, multiplexed stable isotope labelling (TMT 10-plex), and high pH fractionation followed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), redox, PTMs analysis and systems biology analysis.

Tissue sample preparation for mass spectrometry analysis

To compare the pattern of protein alterations produced by I/R, myocardial tissue samples (from the ischemic and remote regions) were collected from the control group (no intervention) and at 20 min, 40 min, 80 min, 2 hours, 6 hours, 12 hours and 24 hours time-points after reperfusion. Samples from the ischemic and remote myocardium of all animals were collected within minutes of euthanasia and processed for proteomics analysis. Protein extracts from the

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homogenized tissue were obtained using the ceramic beads (MagNa Lyser Green Beads apparatus, Roche, Germany) in extraction buffer 50mM Tris-HCl, 1mM EDTA, 1.5% SDS, 50mM iodoacetamide (IAM), pH 8.5. Protein extracts were quantified and pooled accordingly to their concentration for each time-point. We applied a filter-based modification of the GELSILOX technology [77], which allows a simultaneous quantitative analysis of the alterations in the redox state of the Cys sites and of protein abundance [73, 74]. Briefly, proteins were extracted in the presence of an alkylating agent (IAM) that blocks free (reduced) thiol groups. Then, proteins were loaded on a FASP filter (Expedeon, San Diego, CA), which was used as a reaction chamber to 1) wash-away detergents and other contaminants that would hinder tryptic digestion; 2) reduce Cys with dithiothreitol (DTT); 3) alkylate previously oxidized Cys with 50mM methyl methanethiosulfonate (MMTS) and 4) trypsin-digest proteins. Thus, the original redox state of the Cys-containing peptides is determined by the specific mass shift introduced by each alkylating agent in the precursor ion and the fragments. Equal amounts of the resulting peptides were labelled with TMT 10-plex reagents (Thermo Scientific, San Jose, CA, USA).

Peptide fractionation

Labeled peptides were separated into 5 fractions using high pH reversed- phase peptide fractionation (Thermo Scientific) and graded concentrations of acetonitrile (ACN) in triethylamine (0.1%). Desalted and dried peptides were taken up in 300µl of 0.1% trifluoroacetic acid (TFA) solution. The spin column was equilibrated by passing 300µL of ACN twice, followed by 2x300µL of 0.1%

TFA solution. Samples were applied and columns were centrifuged at 3000 x g for 2 minutes. Columns were then washed with 300µL of water. Bound peptides were eluted into 5 fractions with 300 µL of freshly prepared elution solutions: (1) 12.5% ACN, 87.5% triethylamine; (2) 15% ACN, 85% triethylamine; (3) 17.5%

ACN, 82.5% triethylamine; (4) 20% ACN, 80% triethylamine and (5) 50% ACN, 50% triethylamine. Obtained fractions were dried and stored at −20°C until MS analysis.

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Liquid chromatography tandem mass spectrometry (nanoLC- MS/MS)

For shotgun proteomics analysis, the tryptic peptide mixtures were subjected to nanoLC-MS/MS. High-resolution analysis was performed on a nano-HPLC Easy nLC 1000 liquid chromatograph (Thermo Scientific, San Jose, CA, USA) coupled to a QExactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific). Peptides were suspended in 0.1% FA, loaded onto a C18 RP nano-precolumn (Acclaim PepMap100, 75-µm internal diameter, 3-µm particle size and 2-cm length, Thermo Scientific), and separated on an analytical C18 nano-column (EASY-Spray column PepMap RSLC C18, 75-um internal diameter, 3-um particle size and 50-cm length, Thermo Scientific) in a continuous gradient (8–31%B for 240 min, 31–90%B for 2 min, 90%B for 7 min, 90-5%B for 3 min and 2%B for 30 min, where A is 0.1% formic acid in HPLC H2O and B is 90% ACN, 0.1% formic acid in HPLC grade H2O). Spectra were acquired using full ion-scan mode over the mass-to-charge (m/z) range 390–1500 and 70,000 FT-resolution. MS/MS was performed on the top twenty ions in each full MS scan in data-dependent acquisition mode with 45s dynamic exclusion enabled. In label-free experiments, MS data were acquired with a Top10 data‐

dependent MS/MS scan method (topN method) with 30,000 FT-resolution.

Protein identification and quantification

Proteins were identified in the raw files using the SEQUEST HT algorithm integrated in Proteome Discoverer 2.1 (Thermo Finnigan). MS/MS scans were matched against a combined pig and human database (UniProtKB/Swiss-Prot 2017_06 Release). For database searching, parameters were selected as follows:

trypsin digestion with 2 maximum missed cleavage allowed, precursor mass tolerance of 800 ppm, and a fragment mass tolerance of 0.02 ppm. The N- terminal and Lys TMT modifications were chosen as fixed modifications, whereas Met oxidation, Cys carbamidomethylation, and Cys methylthiolation were chosen as variable modifications. The same MS/MS spectra collections were searched against inverted databases constructed from the same target databases.

SEQUEST results were analyzed by the probability ratio method [78]. False

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discovery rate (FDR) was calculated for peptides identified in the inverted database search results using the refined method [79, 80].

Dynamic protein expression profiles after myocardial I/R were characterized using the relative quantification approach by TMT 10-plex stable isotope labeling (SIL) with tandem mass spectrometry (MS/MS). Quantitative information was extracted from MS/MS spectra of TMT-labeled samples using an in-house MSQL library-based R script. The quantitative information from TMT reporter intensities was integrated from the spectrum level to the peptide level and then to the protein level on the basis of the WSPP model [57, 73] using the Generic Integration Algorithm (GIA) [74] . Briefly, for each sample the values = log / were calculated, where is the intensity of the TMT reporter corresponding to sample in the MS/MS spectrum coming from peptide and protein , and is the average intensity of all the TMT reporters from the control samples, which is used as a common reference. The log2-ratio of each peptide ( ) was calculated as the weighted average of its spectra, the protein values ( ) were the weighted average of its peptides, and the grand mean ( ̅) was calculated as the weighted average of all the protein values [73] . The statistical weights of spectra, peptides and proteins ( , and , respectively) and the variances at each one of the three levels ( , , and , respectively), were calculated as described [73] .

The spectrum, peptide and protein variances and the protein values were firstly determined including only non-modified peptides. In a second step, the modified peptides were included in the analysis, which was performed using the variances and protein values calculated previously. For each modified peptide, the standardized variable ( ) was calculated as

= − , >1

where is the number of non-modified peptides with which the corresponding protein was quantified. expresses the deviation between the peptide log2-ratio and the corresponding protein value in units of standard

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deviation. In absence of changes, the distributions of follows very closely the normal distribution (0,1) validating the accuracy of the model.

As input, WSPP uses a list of quantifications in the form of log2-ratios (for example a condition versus control sample) with their statistical weights. From these, WSPP generates the standardized forms of the original variables by computing the quantitative values expressed in units of standard deviation around the means (Zq). For the protein functional analysis, we used the Systems Biology Triangle (SBT) model developed in our group, which estimates functional category averages (Zc) from protein values by performing the protein-to-category integration. To facilitate detection of similar categories (categories sharing many proteins), a clustering algorithm was applied, as described [74].

Label Free (LF) absolute quantification method implemented in MaxQuant proteomics identification and quantitation software [81] was used for the analysis of the label-free proteomics experiments. We express the protein abundances as percentage of the identified proteome, obtained by normalizing the LF intensities of proteins of interest to the sum of all protein intensities.

Protein functional annotation

Quantified proteins were functionally annotated using the Ingenuity Knowledge Database (IPA) [82, 83] and DAVID [84]. The DAVID repository (david.ncifcrf.gov) included 13 functional databases, including Gene Ontology (www.geneontology.org), KEGG (www.genome.jp/kegg), and Panther (www.pantherdb.org), among others.

PTM identification and annotation

For the global PTM analysis, all searches were performed with Comet- PTM as specified before [72] using trypsin digestion without missed cleavages and fixed TMT labeling at N-terminal and Lys (229.162932 Da). Precursor ion tolerance was set to 500 Da. Output files from Comet-PTM were further analyzed using SHIFTS (Systematic Hypothesis-free Identification of modifications with controlled FDR based on ultra-Tolerant database Search) [72]. Bin was set to 0.001 Da for DeltaMass recalibration, and peptide identification filters were set

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at 1% for peak and local FDR and 5% for Global FDR. A Python in-house script was used for semisupervised annotation of the nature of peptide modifications.

The script searched DeltaMass values against Unimod database [85]

(http://www.unimod.org), taking into account the modified amino acid according to Comet-PTM output and also the preceding and consecutive residues, comparing them with the list of amino acids that could be subjected to the modification according to Unimod. If no amino acid was matched, the modification was considered as unassigned and was not taken into account for the analysis. Unexplained DeltaMass values were termed as unknown and were not taken into account for the analysis.

Enrichment analysis

To account for the proteome background, enrichment analysis for PTMs was performed calculating a hypergeometric p-value [86] using the total population of PTMs as the reference population. Biological processes enrichment analysis was performed calculating the hypergeometric p-value [86] for the enrichment of functional categories from GO database [87], using total count of PSMs as frequency value. To account for the contribution of the background cardiac proteome, we used the identified non-modified proteome as reference population.

PCA and HCA analysis

We performed the Principal Components Analysis (PCA) using the ‘stats’

package as part of R [88] and ggplot2 [89] for data visualization. Hierarchical Clustering Analysis (HCA) was performed using R [88]. First, we computed the distance matrix for all modified peptides applying the ‘factoextra’ package [90] in R and using Pearson correlation coefficient as distance measure. Then, we ordered the correlation matrix using the hierarchical clustering algorithm in the

‘corrplot’ [91] R package.

Immunoblotting

Western blot was performed according to standard protocols. Briefly, 10μg of heart tissue extracts were loaded per lane separated on the 4-10%

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polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) (Immobilon-FL, Milipore) membranes for fluorescence applications. Results were obtained using the 4-hydroxynonenal (4-HNE, Abcam, ab46545) antibodies. Secondary antibodies goat anti-mouse IgG DyLight 800 (Rockland, 610-145-121) and goat anti-rabbit IgG Alexa Fluor 680 (Thermo Fisher Scientific, A-21076) were used against the corresponding primary antibodies and the images were acquired with the ODYSSEY Infrared Imaging System (LI-COR).

Myeloperoxidase activity detection

MPO activity was measured as an indicator of neutrophil infiltration in the ischaemic myocardium using a Myeloperoxidase Colorimetric Activity Assay Kit (Abcam, ab105136) according to the manufacturer’s instructions. One unit of MPO is defined as the amount of MPO that hydrolyzes the substrate and generates taurine chloramine to consume 1 mmol trinitrobenzene (TNB) per min at 25°C.

Assays for Oxidative Stress Measurement

Malondialdehyde (MDA)

Cardiac tissue homogenates were assessed for the presence of lipid peroxidation product (MDA presence) (Abcam; ab118970) according to the manufacturer’s instructions. Briefly, ischemic tissue from pig at different times after I/R were collected in 303µL of MDA lysis solution using a TissueLyser LT (Qiagen, USA) homogenizer precooled on ice. Samples were then centrifuged at 13.000 x g for 10 min and total protein content was quantified using RCDC Assay (BIORAD). To generate MDA-TBA adduct, 600µL of TBA reagent was added into each vial containing the supernatant previously collected. Samples were then incubated at 95°C for 60 min and subsequently cooled in an ice bath for 10 min.

Each 800µl of TBA/sample and standard mix (for the standard curve calculation) were added in a 96-wells plate (BD science). The absorbance of TBA-MDA adduct was measured at 532 nm. The MDA concentration in standard and samples was determined from their absorbance as per the manufacturer’s instructions.

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50 H2O2 Measurement

H2O2 production was measured with an Amplex Red H2O2 assay kit (Molecular Probes; Invitrogen) according to the manufacturer’s instructions. In brief, left ventricular blocks (30–50mg) were incubated with Amplex Red (100µmol/L) and horseradish peroxidase (1 U/mL) for 30 min at 37°C in Krebs–

HEPES buffer protected from light. The supernatant was then transferred to a 96-well plate, and absorbance was measured (560 nm). Background fluorescence, determined in a control reaction without sample, was subtracted from each value. H2O2 release was calculated using H2O2 standards and expressed as micromoles per milligram of tissue.

Statistical and Data Analysis

Western blot analysis to determine significant differences between groups after densitometry analysis and statistical analysis of label free proteomics, enzymatic and biochemical assays were performed using the Mann-Whitney test, using the GraphPad Prism 7.02 software. The statistical differences in the peptide and protein quantitative values (Zp and Zq, respectively) between all I/R groups as well as the results of all experiments for nonreperfused and preconditioned experimental groups were evaluated by Kruskal-Wallis test. For the assessment of changes in the individual oxidatively modified peptides in the neutrophil depletion experiments we employed multiple t tests (P value <0.05) with Benjamini and Hochberg’s FDR method post hoc correction (q value <0.05). For the experiments in pig model the threshold was set to P value <0.01 and q value

<0.01.

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Results

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Early reperfusion induces two temporally separated oxidative episodes in the ischemic tissue

Previous Cys-redox proteomics study in a pig model of I/R at 2h, 24h, 4d and 7d after reperfusion showed an increase in Cys reversible oxidation peaking at 24h and already increased as early as 2h after reperfusion [66]. This results as well as the evidence in growing body of research in the area of early reperfusion- induced oxidation [92-100] led us to design a detailed timeline from the earliest moments (20min) up to 24h after reperfusion time in a pig model of I/R (Figure 5).

Figure 5 Study design. Pig model of ischemia reperfusion (I/R). The study population comprised 10 groups of pigs (n=4/group). Groups 1 to 8 were used for the analysis of redox, PTMs and protein expression changes during the first 24h after I/R in remote and ischemic tissue. Closed-chest 40min ischemia followed by reperfusion was performed and pigs sacrificed at 20min, 40min, 80min, 2h, 6h, 12h and 24h after reperfusion. Additional four sacrificed healthy pigs served as controls. CMR (cardiac magnetic resonance) scans were acquired by Borja Ibañez’s group at CNIC (data not shown).

This new experimental setup allowed us to gain unique insights into the oxidative events taking place in the lesion area. The analysis of the dynamic thiol redox proteome allowed us to identify 491 oxidized and 1680 reduced Cys.

Interestingly, this analysis revealed an increase in Cys-reversible oxidation (Figure 6).

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Results

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Figure 6 Reversible thiol redox alterations in the ischemic tissue during the first 24 hours after MI. Oxidized Cys-peptides changes along time course. (a) Total population of oxidized cysteines (491 peptides) Zp peptide values are reported as the log2-fold changes in each condition with respect to controls. (b) Zp averages are reported as the complete peptide population average value at each time point for each individual animal. ns indicates p value > 0.05, * Indicates p value < 0.05, *** indicates p value<0.001. P-values are calculated using Kruskal-Wallis test. Full list of oxidized and reduced Cys- containing peptides can be found in Supplementary Table S1.

The complete Cys population (Supplementary Table S1) of oxidized-Cys- containing peptides quantitative values (Zp; log2 FC expressed in SD units) for each I/R time point with respect to the control group was subjected to hierarchical clustering analysis (HCA), revealing a subset of oxidized Cys population that significantly increase during 2h-24h after I/R (Figure 7, Supplementary Table S1, marked in red). This observation confirmed the previously mentioned maximal increase in Cys oxidation after 24h post-MI.

The high correlation of Zp values along the reperfusion time indicates that reversible oxidation affects the same peptide sequences (Figure 8). The experimental group of 6h reperfusion time had the highest correlation (R²>0.7) with 3 other time points (2h, R²=0.736; 12h, R²=0.843; 24h, R²=0.883), suggesting a focal time segment (2h to 24h) in the increase of reversible Cys oxidation (Figure 8).

a

0 5 10 15

20’ 40’ 80’ 2h 6h 12h 24h

-10 -5

Zp distribution

Zp > 1.5

Zp < -1.5 -0.5

0.0 0.5 1.0 1.5 2.0

20' 40' 80' 2h 6h 12h 24h

ns

***

ns

*

Zp average

Kruskal-Wallis, p = 0.0223

Unraveling the dynamic oxidative processes after ischemia/reperfusion in the heart using new proteomics approaches

Facultad de Medicina Departamento de Bioquímica